Ghrh or analogues thereof for use in treatment of hepatic disease

ABSTRACT

The present application relates to novel methods for preventing, slowing the progression of, or treating nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), and/or liver fibrosis, and/or reducing the risks of liver cancer in subjects, such as HIV-infected subjects, using a GHRH molecule, e.g., trans-3-hexenoyl-GHRH(1-44)-NH2, or a pharmaceutically acceptable salt thereof. The subjects may have particular pathological features such as liver fibrosis, a hepatic fat fraction (HFF) of at least about 10%, serum alanine aminotransferase (ALT) levels of at least about 30 U/L, and/or a NAFLD Activity Score (NAS) of at least 4 or 5.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 17/018,878, filed on Sep. 11, 2020, which is a continuation ofU.S. patent application Ser. No. 16/832,128, filed on Mar. 27, 2020, nowU.S. Pat. No. 10,799,562, which claims the benefit of U.S. Provisionalapplication No. 62/826,284, filed on Mar. 29, 2019, and of U.S.Provisional application No. 62/861,187, filed on Jun. 13, 2019, whichare incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under NationalInstitutes of Health, National Institute of Allergy and InfectiousDiseases Grant No. U01 A1115711. The Government has certain rights inthis invention.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable formentitled “11718_361_SeqList.txt”, created on Mar. 25, 2020 and having asize of about 5 KB. The computer readable form is incorporated herein byreference.

TECHNICAL FIELD

The present disclosure generally relates to the management of liverdisease, such as nonalcoholic fatty liver (NAFL), nonalcoholicsteatohepatitis (NASH), and liver fibrosis.

BACKGROUND ART

NAFL is defined by excess storage of triglyceride in hepatocytes(steatosis) and is often characterized by resultant inflammation,cellular ballooning and damage, and fibrosis. Significant changes inthis regard lead to NASH. Nonalcoholic fatty liver disease (NAFLD) mayprogress to fibrosis and ultimately cirrhosis and is an increasinglyimportant cause of end-stage liver disease in the general population,and has also been studied in people living with HIV (PLWH; Rockstroh JK. Curr HIV/AIDS Rep 2017; 14:47-53; Vodkin I, et al. Aliment PharmacolTher 2015; 41:368-78). NAFL/NASH have a higher prevalence in HIVpatients and tend to progress faster than in the general population. Incontrast to many HIV-associated comorbidities that worsen with increasedHIV-disease severity, NAFLD may occur more commonly in HIV patients withhigher CD4⁺ T-cell counts and weight gain and it is associated withcentral adiposity (Guaraldi G, et al. Clin Infect Dis 2008; 47:250-7;van der Poorten D, et al. Hepatology 2008; 48:449-57; Maurice J B, etal. AIDS 2017; 31:1621-32). In PLWH, weight gain, abdominal fataccumulation, and increases in visceral fat are common and seen evenwith newer antiretrovirals (Lake J E, et al. Clin Infect Dis 2017;64:1422-9). Although Vitamin E (Sanyal A J. N Engl J Med 2010;362:1675-85; Lavine J E, et al. JAMA 2011; 305:1659-68; Sato K, et al.Nutrition 2015; 31:923-30) and pioglitazone⁷ have been shown to improvehistological features of NASH in the general population, there are noproven pharmacologic therapies for NAFLD or NASH in people living withHIV. One product, aramchol (an oral stearoyl-coenzyme-A-desaturase-1inhibitor known to reduce hepatic-fat content in patients with primaryNAFLD), was assessed in patients with HIV-associated NAFLD and it had nosignificant effect in this specific patient population (Ajmera et al.,Hepatology. 2019 Apr. 23. doi: 10.1002/hep.30674. [Epub ahead ofprint]).

Liver fibrosis results from chronic damage to the liver in conjunctionwith the accumulation of extracellular matrix (ECM) proteins, which is acharacteristic of most types of chronic liver diseases. Advanced liverfibrosis results in cirrhosis, liver failure, and portal hypertensionand often requires liver transplantation. There is currently noeffective therapy that effectively target liver fibrosis associated withNAFL/NASH.

There is thus a need for novel therapies for the management of NAFLD,NASH and/or liver fibrosis, notably in HIV-infected patients.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE DISCLOSURE

The present disclosure generally relates to the management of liverdisease, such as nonalcoholic fatty liver disease (NAFLD). NAFLD refersto a fatty liver that is not related to alcohol use, and is furtherdivided into nonalcoholic fatty liver (NAFL) and nonalcoholicsteatohepatitis (NASH) with or without fibrosis, and in an aspectrelates to the use of growth hormones (GH) secretagogues, and morespecifically to Growth Hormone-Releasing Hormone (GHRH) or analogsthereof for use in the management (including prevention and/ortreatment) of such liver disease or related conditions.

In various aspects and embodiments, the present disclosure provides thefollowing items:

1. A method for preventing or treating nonalcoholic fatty liver (NAFL)or nonalcoholic steatohepatitis (NASH) in a subject in need thereofcomprising administering an effective amount oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof to said subject, wherein said subject has (i) a hepatic fatfraction (HFF) of at least about 5% or 10% as measured by protonmagnetic resonance spectroscopy (¹H MRS) (or any other suitable method;(ii) serum alanine aminotransferase (ALT) levels of at least about 25 or30 U/L; (iii) a NAFLD Activity Score (NAS) of at least 3, 4 or 5 asmeasured by the NAS Clinical Research Network (NAS CRN) scoring system;(iv) liver fibrosis or (iv) any combination of (i) to (iv).2. The method of item 1, wherein said subject has an HFF of at leastabout 5% or 10%.3. The method of item 1, wherein said subject has an HFF of at leastabout 15%.4. The method of item 1, wherein said subject has an HFF of at leastabout 20%.5. The method of any one of items 1 to 4, wherein said subject has serumALT levels of at least about 25 or 30 U/L.6. The method of any one of items 1 to 4, wherein said subject has serumALT levels of at least about 35 U/L.7. The method of any one of items 1 to 6, wherein said subject has a NASof at least 6.8. The method of item 7, wherein said subject has a NAS of at least 7.9. The method of any one of items 1 to 8, wherein said subject has liverfibrosis, for example stage 1A liver fibrosis or worse, preferably stage1C or stage 2 liver fibrosis or worse.10. The method of any one of items 1 to 9, wherein said subject suffersfrom human immunodeficiency virus (HIV) infection.11. The method of any one of items 1 to 10, wherein said subject has abody mass index (BMI) of at least about 25.12. The method of item 11, wherein said subject has a BMI of at leastabout 30.13. The method of any one of items 1 to 12, wherein said methodcomprises administering an effective amount of a pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ to said subject.14. The method of item 13, wherein said pharmaceutically acceptable saltof trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.15. The method of any one of items 1 to 14, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered at a daily dose of about 1 mg to about 4 mg.16. The method of item 15, wherein said trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂or pharmaceutically acceptable salt thereof is administered at a dailydose of about 1 mg to about 2 mg.17. The method of any one of items 1 to 16, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered by subcutaneous injection.18. The method of any one of items 1 to 17, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.19. The method of item 18, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.20. The method of item 18, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.21. Trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptablesalt thereof for use in the prevention or treatment of nonalcoholicfatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) in a subject,wherein said subject has (i) a hepatic fat fraction (HFF) of at leastabout 5 or 10% as measured by proton magnetic resonance spectroscopy (¹HMRS); (ii) serum alanine aminotransferase (ALT) levels of at least about25 or 30 U/L; (iii) a NAFLD Activity Score (NAS) of at least 5 asmeasured by the NAS Clinical Research Network (NAS CRN) scoring system;or (iv) any combination of (i) to (iii).22. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 21, wherein said subject has anHFF of at least about 5 or 10%.23. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 21, wherein said subject has anHFF of at least about 15%.24. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 21, wherein said subject has anHFF of at least about 20%.25. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 24, whereinsaid subject has serum ALT levels of at least about 25 or 30 U/L.26. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 24, whereinsaid subject has serum ALT levels of at least about 35 U/L.27. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 26, whereinsaid subject has a NAS of at least 6.28. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 27, wherein said subject has aNAS of at least 7.29. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 28, whereinsaid subject has liver fibrosis, for example stage 1A liver fibrosis orworse, preferably stage 1C or stage 2 liver fibrosis or worse.30. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 29, whereinsaid subject suffers from human immunodeficiency (HIV) infection.31. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 31, whereinsaid subject has a body mass index (BMI) of at least about 25.32. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 31, wherein said subject has aBMI of at least about 30.33. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 33, wherein apharmaceutically acceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ isused.34. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 33, wherein said pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.35. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 34, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 4mg.36. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 37, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 2mg.37. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 36, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration by subcutaneous injection.38. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21 to 37, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.39. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 38, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.40. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 38, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.41. A method for (i) preventing or reducing the development orprogression of liver fibrosis or (ii) reducing liver fibrosis in asubject, comprising administering an effective amount oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof to said subject.42. The method of item 41, wherein said subject has a fibrosis score ofat least 10 prior to the treatment.43. The method of item 41, wherein said subject has a fibrosis score ofat least 2 prior to the treatment.44. The method of any one of items 41 to 43, wherein said subject has anHFF of at least about 10%.45. The method of item 44, wherein said subject has an HFF of at leastabout 15%.46. The method of item 45, wherein said subject has an HFF of at leastabout 20%.47. The method of any one of items 41 to 46, wherein said subject hasserum ALT levels of at least about 30 U/L.48. The method of any one of items 41 to 46, wherein said subject hasserum ALT levels of at least about 35 U/L.49. The method of any one of items 41 to 48, wherein said subject has aNAFLD Activity Score (NAS) of at least 2 as measured by the NAS ClinicalResearch Network (NAS CRN) scoring system.50. The method of item 49, wherein said subject has a NAS of at least 3.51. The method of any one of items 41 to 50, wherein said subjectsuffers from human immunodeficiency virus (HIV) infection.52. The method of any one of items 41 to 51, wherein said subject has abody mass index (BM I) of at least about 25.53. The method of item 52, wherein said subject has a BMI of at leastabout 30.54. The method of any one of items 41 to 53, wherein said methodcomprises administering an effective amount of a pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ to said subject.55. The method of item 54, wherein said pharmaceutically acceptable saltof trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.56. The method of any one of items 41 to 55, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered at a daily dose of about 1 mg to about 4 mg.57. The method of item 56, wherein said trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂or pharmaceutically acceptable salt thereof is administered at a dailydose of about 1 mg to about 2 mg.58. The method of any one of items 41 to 57, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered by subcutaneous injection.59. The method of any one of items 41 to 58, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.60. The method of item 59, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.61. The method of item 59, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.62. Trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptablesalt thereof for use in (i) preventing or reducing the development orprogression of liver fibrosis or (ii) reducing liver fibrosis in asubject.63. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 62, wherein said subject has afibrosis score of at least 10 prior to the treatment.64. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 62, wherein said subject has afibrosis score of at least 2 prior to the treatment.65. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 64, whereinsaid subject has an HFF of at least about 10%.66. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 65, wherein said subject has anHFF of at least about 15%.67. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 65, wherein said subject has anHFF of at least about 20%.68. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 67, whereinsaid subject has serum ALT levels of at least about 30 U/L.69. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 67, whereinsaid subject has serum ALT levels of at least about 35 U/L.70. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 69, whereinsaid subject has a NAFLD Activity Score (NAS) of at least 2 as measuredby the NAS Clinical Research Network (NAS CRN) scoring system.71. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 70, wherein said subject has aNAS of at least 3.72. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 71, whereinsaid subject suffers from human immunodeficiency (HIV) infection.73. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 72, whereinsaid subject has a body mass index (BMI) of at least about 25.74. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 73, wherein said subject has aBMI of at least about 30.75. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 74, wherein apharmaceutically acceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ isused.76. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 75, wherein said pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.77. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 76, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 4mg.78. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 77, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 2mg.79. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 78, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration by subcutaneous injection.80. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 62 to 79, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.81. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 80, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.82. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 80, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.83. The method of any one of items 1-20 and 41-61, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered for a period of at least 3, 6 or 9 months.84. The method of item 83, wherein said trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂or pharmaceutically acceptable salt thereof is administered for a periodof at least 12 months.85. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 21-40 and 62-82,wherein said trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceuticallyacceptable salt thereof is used for a period of at least 3, 6 or 9months.86. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 85, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is used for a period of at least 12 months.87. A method for reducing the risk of developing liver cancer in asubject suffering from nonalcoholic fatty liver (NAFL) or nonalcoholicsteatohepatitis (NASH) comprising administering an effective amount oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof to said subject.88. The method of item 87, wherein said method comprises administeringan effective amount of a pharmaceutically acceptable salt oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ to said subject.89. The method of item 88, wherein said pharmaceutically acceptable saltof trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.90. The method of any one of items 87 to 89, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered at a daily dose of about 1 mg to about 4 mg.91. The method of item 90, wherein said trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂or pharmaceutically acceptable salt thereof is administered at a dailydose of about 1 mg to about 2 mg.92. The method of any one of items 87 to 91, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered by subcutaneous injection.93. The method of any one of items 87 to 92, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.94. The method of item 93, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.95. The method of item 93, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.96. The method of any one of items 87 to 95, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered for a period of at least 3, 6 or 9 months.97. The method of item 96, wherein said trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂or pharmaceutically acceptable salt thereof is administered for a periodof at least 12 months.98. Trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptablesalt thereof for use in reducing the risk of developing liver cancer ina subject suffering from nonalcoholic fatty liver (NAFL) or nonalcoholicsteatohepatitis (NASH).99. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 98, wherein a pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is used.100. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 99, wherein said pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.101. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 98 to 100, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 4mg.102. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 101, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 2mg.103. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 98 to 102, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration by subcutaneous injection.104. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 98 to 103, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.105. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 104, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.106. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 105, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.107. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to any one of items 98 to 106, whereinsaid trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is used for a period of at least 3, 6 or 9 months.108. The trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptablesalt thereof for use according to item 107, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is used for a period of at least 12 months.109. Use of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof for the prevention or treatment of nonalcoholicfatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) in a subject,wherein said subject has (i) a hepatic fat fraction (HFF) of at leastabout 5 or 10% as measured by proton magnetic resonance spectroscopy (¹HMRS); (ii) serum alanine aminotransferase (ALT) levels of at least about25 or 30 U/L; (iii) a NAFLD Activity Score (NAS) of at least 5 asmeasured by the NAS Clinical Research Network (NAS CRN) scoring system;or (iv) any combination of (i) to (iii).110. Use of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof for the preparation of a medicament for theprevention or treatment of nonalcoholic fatty liver (NAFL) ornonalcoholic steatohepatitis (NASH) in a subject, wherein said subjecthas (i) a hepatic fat fraction (HFF) of at least about 5 or 10% asmeasured by proton magnetic resonance spectroscopy (¹H MRS); (ii) serumalanine aminotransferase (ALT) levels of at least about 25 or 30 U/L;(iii) a NAFLD Activity Score (NAS) of at least 5 as measured by the NASClinical Research Network (NAS CRN) scoring system; or (iv) anycombination of (i) to (iii).111. The use according to item 109 or 110, wherein said subject has anHFF of at least about 5 or 10%.

112. The use according to item 109 or 110, wherein said subject has anHFF of at least about 15%.

113. The use according to item 109 or 110, wherein said subject has anHFF of at least about 20%.114. The use according to any one of items 109 to 113, wherein saidsubject has serum ALT levels of at least about 25 or 30 U/L.115. The use according to any one of items 109 to 114, wherein saidsubject has serum ALT levels of at least about 35 U/L.116. The use according to any one of items 109 to 115, wherein saidsubject has a NAS of at least 6.117. The use according to item 116, wherein said subject has a NAS of atleast 7.118. The use according to any one of items 109 to 117, wherein saidsubject has liver fibrosis, for example stage 1A liver fibrosis orworse, preferably stage 10 or stage 2 liver fibrosis or worse.119. The use according to any one of items 109 to 118, wherein saidsubject suffers from human immunodeficiency (HIV) infection.120. The use according to any one of items 109 to 119, wherein saidsubject has a body mass index (BMI) of at least about 25.121. The use according to item 120, wherein said subject has a BMI of atleast about 30.122. The use according to any one of items 109 to 121, wherein apharmaceutically acceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ isused.123. The use according to item 122, wherein said pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.124. The use according to any one of items 109 to 123, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 4mg.125. The use according to item 124, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 2mg.126. The use according to any one of items 109 to 125, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration by subcutaneous injection.127. The use according to any one of items 109 to 126, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.128. The use according to item 127, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.129. The use according to item 127 or 128, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₁₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.130. Use of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof for (i) preventing or reducing the developmentor progression of liver fibrosis or (ii) reducing liver fibrosis in asubject.131. Use of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof for the preparation of a medicament for (i)preventing or reducing the development or progression of liver fibrosisor (ii) reducing liver fibrosis in a subject.132. The use according to item 130 or 131, wherein said subject has afibrosis score of at least 10 prior to the treatment.133. The use according to item 130 or 131, wherein said subject has afibrosis score of at least 2 prior to the treatment.134. The use according to any one of items 130 to 133, wherein saidsubject has an HFF of at least about 10%.135. The use according to item 134, wherein said subject has an HFF ofat least about 15%.136. The use according to item 135, wherein said subject has an HFF ofat least about 20%.137. The use according to any one of items 130 to 136, wherein saidsubject has serum ALT levels of at least about 30 U/L.138. The use according to any one of items 130 to 137, wherein saidsubject has serum ALT levels of at least about 35 U/L.139. The use according to any one of items 130 to 138, wherein saidsubject has a NAFLD Activity Score (NAS) of at least 2 as measured bythe NAS Clinical Research Network (NAS CRN) scoring system.140. The use according to item 139, wherein said subject has a NAS of atleast 3.141. The use according to any one of items 130 to 140, wherein saidsubject suffers from human immunodeficiency (HIV) infection.142. The use according to any one of items 130 to 141, wherein saidsubject has a body mass index (BMI) of at least about 25.143. The use according to item 142, wherein said subject has a BMI of atleast about 30.144. The use according to any one of items 130 to 143, wherein apharmaceutically acceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ isused.145. The use according to item 144, wherein said pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.146. The use according to any one of items 130 to 145, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 4mg.147. The use according to item 146, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 2mg.148. The use according to any one of items 130 to 147, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration by subcutaneous injection.149. The use according to any one of items 130 to 148, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.150. The use according to item 149, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.151. The use according to item 149 or 150, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.152. Use of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof for reducing the risk of developing liver cancerin a subject suffering from nonalcoholic fatty liver (NAFL) ornonalcoholic steatohepatitis (NASH).153. Use of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof for the preparation of a medicament for reducingthe risk of developing liver cancer in a subject suffering fromnonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH).154. The use according to item 152 or 153, wherein a pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is used.155. The use according to item 154, wherein said pharmaceuticallyacceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ is an acetate salt.156. The use according to any one of items 152 to 155, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 4mg.157. The use according to item 156, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration at a daily dose of about 1 mg to about 2mg.158. The use according to any one of items 152 to 157, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for administration by subcutaneous injection.159. The use according to any one of items 152 to 158, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising at leastone pharmaceutically acceptable excipient.160. The use according to item 159, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 10mg/mL.161. The use according to item 159 or 160, wherein the concentration oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof in the pharmaceutical composition is about 1 mg/mL to about 8mg/mL.162. The use according to any one of items 109 to 161, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for use for a period of at least 3, 6 or 9 months.163. The use according to item 162, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is for use for a period of at least 12 months.

Other objects, advantages and features of the present disclosure willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

In the appended drawings:

FIG. 1 is a diagram showing the participant flow and reasons for patientexclusion from the study.

FIG. 2 is a table showing the effects of tesamorelin on hepatic fat,metabolic, and immunologic indices.

FIGS. 3A and 3B are graphs depicting the change in absolute (FIG. 3A)and relative (FIG. 3B) liver fat content between baseline and 12 months,with p-values shown for t-test comparing change between groups.

FIG. 3C is a graph depicting the percent resolution of steatosis,defined as 12-month hepatic fat fraction <5%, with P-value for PearsonChi-Square.

FIG. 3D is a graph depicting the change in alanine aminotransferase(ALT) between baseline and 12 months for those with ALT 30 U/L atbaseline, with p-value for t-test comparing change between groups.

FIG. 4A is a graph depicting the percentage of patients with progressionof fibrosis at 12 months, with P-value for Pearson Chi-Square.

FIG. 4B is a graph depicting the relationship between change in fibrosisand change in NAS score at 12 months, with P-value for ANOVA.

FIG. 5 shows the structure of tesamorelin(trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂; SEQ ID NO: 1).

DETAILED DESCRIPTION

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the subject matter (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext.

The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All subsets of values within the ranges arealso incorporated into the specification as if they were individuallyrecited herein.

Similarly, herein a general chemical structure with various substituentsand various radicals enumerated for these substituents is intended toserve as a shorthand method of referring individually to each and everymolecule obtained by the combination of any of the radicals for any ofthe substituents. Each individual molecule is incorporated into thespecification as if it were individually recited herein. Further, allsubsets of molecules within the general chemical structures are alsoincorporated into the specification as if they were individually recitedherein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (“e.g.”, “suchas”) provided herein, is intended merely to better illustrate thedisclosure and does not pose a limitation on the scope of the disclosureunless otherwise claimed.

Herein, the term “about” has its ordinary meaning. The term “about” isused to indicate that a value includes an inherent variation of errorfor the device or the method being employed to determine the value, orencompass values close to the recited values, for example within 10% ofthe recited values (or range of values).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

In the studies described herein, it is shown that administration oftesamorelin (trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂, acetate salt)significantly reduces and/or normalizes liver fat content and preventsprogression of liver fibrosis in HIV-infected subjects with NAFL/NASHwith or without fibrosis. The results provide evidence that thebeneficial effects of tesamorelin treatment are more prominent insubjects having high NAFLD Activity Score (NAS) prior to treatment, i.e.subjects having more advanced or severe disease. In view of the factthat the fibrosis stage is the strongest predictor of mortality inpatients with NAFLD and that a high percentage of HIV patients withNAFLD demonstrate progression of fibrosis over 1 year, the prevention ofliver fibrosis by tesamorelin treatment in patients with high NAS shownin the present study is clinically important. Furthermore, Tesamorelinwas unexpectedly shown to have a more pronounced effect on the reductionof liver fat relative to visceral fat. Gene expression analyses revealedthat administration of tesamorelin was associated with upregulation ofgenes pertaining to oxidative phosphorylation and genes associated withgood prognosis of hepatocellular carcinoma (HCC), and down-regulation ofgenes pertaining to hepatic inflammation, tissue repair, cell turnover,and poor prognosis of HCC.

In an aspect, the present disclosure provides a method for treating NAFLor NASH with or without fibrosis in a subject in need thereof comprisingadministering an effective amount of a GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof, to said subject, wherein said subject has (i) a hepatic fatfraction (HFF) of at least 5, 6, 7, 8, 9 or 10% (for example as measuredby proton magnetic resonance spectroscopy (¹H MRS)); (ii) serum alanineaminotransferase (ALT) levels of at least 25, 26, 27, 28, 29 or 30 U/L;(iii) a NAS of at least 1, 2, 3, 4 or 5, preferably at least 3, 4 or 5,as measured by the NAS Clinical Research Network (NAS CRN) scoringsystem; (iv) liver fibrosis, e.g., stage 1A, 1B, 1C, 2, 3 liver fibrosisor worse; or (v) any combination of (i) to (iv).

In another aspect, the present disclosure provides a method for treatingNAFL or NASH in a subject having a hepatic fat fraction (HFF) of atleast about 5 or 10% (for example as measured by proton magneticresonance spectroscopy (¹H MRS)), said method comprising administeringan effective amount of a GHRH molecule or a pharmaceutically acceptablesalt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or apharmaceutically acceptable salt thereof, to said subject. The presentdisclosure also provides the use of a GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof, for treating NAFL or NASH in a subject having a HFF of at leastabout 5 or 10% (for example as measured by ¹H MRS). The presentdisclosure also provides the use of a GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof, for the manufacture of a medicament for treating NAFL or NASHin a subject having a HFF of at least about 5 or 10% (for example asmeasured ¹H MRS). The present disclosure also provides a GHRH moleculeor a pharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof, for use in the treatment of NAFL or NASH in a subject having aHFF of at least about 5 or 10% (for example as measured by protonmagnetic resonance spectroscopy (¹H MRS)).

In another aspect, the present disclosure provides a method for treatingNAFL or NASH in a subject having serum ALT levels at least about 25 or30 U/L, said method comprising administering an effective amount of aGHRH molecule or a pharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof, to said subject. The present disclosure also provides the useof a GHRH molecule or a pharmaceutically acceptable salt thereof,preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof, for treating NAFL or NASH in a subject havingserum ALT levels at least about 25 or 30 U/L. The present disclosurealso provides the use of a GHRH molecule or a pharmaceuticallyacceptable salt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for treating NAFL or NASH in a subject having serum ALTlevels at least about 25 or 30 U/L. The present disclosure also providesa GHRH molecule or a pharmaceutically acceptable salt thereof,preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof, for use in the treatment of NAFL or NASH in asubject having serum ALT levels at least about 25 or 30 U/L.

In another aspect, the present disclosure provides a method for (i)preventing or reducing the development or progression of liver fibrosisor (ii) reducing liver fibrosis in a subject suffering from NAFL or NASHwith or without fibrosis, said method comprising administering aneffective amount of a GHRH molecule or a pharmaceutically acceptablesalt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or apharmaceutically acceptable salt thereof, to said subject. The presentdisclosure also provides the use of a GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof, for (i) preventing or reducing the development or progressionof liver fibrosis or (ii) reducing liver fibrosis in a subject sufferingfrom NAFL or NASH with or without fibrosis. The present disclosure alsoprovides the use of a GHRH molecule or a pharmaceutically acceptablesalt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for (i) preventing or reducing the development or progressionof liver fibrosis or (ii) reducing liver fibrosis in a subject sufferingfrom NAFL or NASH with or without fibrosis. The present disclosure alsoprovides a GHRH molecule or a pharmaceutically acceptable salt thereof,preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof, for use in (i) preventing the development orprogression of liver fibrosis or (ii) reducing liver fibrosis in asubject suffering from NAFL or NASH with or without fibrosis.

In another aspect, the present disclosure provides a method formaintaining or reducing the NAS in a subject suffering from NAFL or NASHand having a NAS of at least 1, 2, 3, 4 or 5, preferably at least 3, 4or 5, as measured by the NAS CRN scoring system, said method comprisingadministering an effective amount of a GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof, to said subject. The present disclosure also provides the useof a GHRH molecule or a pharmaceutically acceptable salt thereof,preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceuticallyacceptable salt thereof, for maintaining or reducing the NAS in asubject suffering from NAFL or NASH with or without fibrosis and havinga NAS of at least 1, 2, 3, 4 or 5, preferably at least 3, 4 or 5, asmeasured by the NAS CRN scoring system. The present disclosure alsoprovides the use of a GHRH molecule or a pharmaceutically acceptablesalt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for maintaining or reducing the NAS in a subject sufferingfrom NAFL or NASH with or without fibrosis and having a NAS of at least1, 2, 3, 4 or 5, preferably at least 3, 4 or 5, as measured by the NASCRN scoring system. The present disclosure also provides a GHRH moleculeor a pharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof, for use in maintaining or reducing the NAS in a subjectsuffering from NAFL or NASH with or without fibrosis and having a NAS ofat least 1, 2, 3, 4 or 5, preferably at least 3, 4 or 5, as measured bythe NAS CRN scoring system.

In another aspect, the present disclosure provides a method for reducingthe risk or likelihood of developing liver cancer in a subject sufferingfrom NAFL or NASH comprising administering an effective amount oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof to said subject. The present disclosure also providesTrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof for reducing the risk or likelihood of developing liver cancerin a subject suffering from NAFL or NASH. The present disclosure alsoprovides the use of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or apharmaceutically acceptable salt thereof for reducing the risk orlikelihood of developing liver cancer in a subject suffering from NAFLor NASH, or for the manufacture of a medicament for reducing the risk orlikelihood of developing liver cancer in a subject suffering from NAFLor NASH. In an embodiment, the liver cancer is hepatocellular carcinoma(HCC).

In an embodiment, the above-mentioned subject has an HFF of at leastabout 5, 6, 7, 8, 9 or 10% as measured for example by ¹H MRS. In anembodiment, the above-mentioned subject has an HFF of at least about 11%as measured for example by ¹H MRS. In an embodiment, the subject has anHFF of at least about 12% as measured for example by ¹H MRS. In anembodiment, the subject has an HFF of at least about 13% as measured forexample by ¹H MRS. In an embodiment, the subject has an HFF of at leastabout 14% as measured for example by ¹H MRS. In an embodiment, thesubject has an HFF of at least about 15% as measured for example by ¹HMRS. In an embodiment, the subject has an HFF of at least about 16% asmeasured for example by ¹H MRS. In an embodiment, the subject has an HFFof at least about 17% as measured for example by ¹H MRS. In anembodiment, the subject has an HFF of at least about 18% as measured forexample by ¹H MRS. In an embodiment, the subject has an HFF of at leastabout 19% as measured for example by ¹H MRS. In an embodiment, thesubject has an HFF of at least about 20% as measured for example by ¹HMRS. In an embodiment, the subject has an HFF of at least about 21% asmeasured for example by ¹H MRS. In an embodiment, the subject has an HFFof at least about 22% as measured for example by ¹H MRS. In anembodiment, the subject has an HFF of at least about 23% as measured forexample by ¹H MRS. In an embodiment, the subject has an HFF of at leastabout 24% as measured for example by ¹H MRS. In an embodiment, thesubject has an HFF of at least about 25% as measured for example by ¹HMRS. In an embodiment, the subject has an HFF of at least about 30% asmeasured for example by ¹H MRS. In an embodiment, the subject has an HFFof at least about 35% as measured for example by ¹H MRS.

In an embodiment, the above-mentioned method or use comprisesidentifying a subject having an HFF of at least about 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, or35%, as measured for example by ¹H MRS.

In an embodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, results in no significant change (i.e. stabilization ornormalization), or a reduction, of HFF.

In another embodiment, the administration of the GHRH molecule orapharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least about 35% (relative reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least about 40% (relative reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least about 45% (relative reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least about 50% (relative reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least two-fold. In an embodiment, theadministration of the GHRH molecule or a pharmaceutically acceptablesalt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ orpharmaceutically acceptable salt thereof, reduces HFF by at leastthree-fold. In an embodiment, the administration of the GHRH molecule ora pharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least 4-fold. In an embodiment, theadministration of the GHRH molecule or a pharmaceutically acceptablesalt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ orpharmaceutically acceptable salt thereof, reduces HFF by at least5-fold.

In an embodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least 4% or 5% (absolute reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least 6% (absolute reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least 7% (absolute reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least 8% (absolute reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least 9% (absolute reduction). In anembodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces HFF by at least 10% (absolute reduction).

In an embodiment, the above-mentioned subject has a NAS of at least 1 asmeasured by the NAS CRN scoring system. In an embodiment, theabove-mentioned subject has a NAS of at least 2 as measured by the NASCRN scoring system. In an embodiment, the above-mentioned subject has aNAS of at least 3 as measured by the NAS CRN scoring system. In anembodiment, the above-mentioned subject has a NAS of at least 4 asmeasured by the NAS CRN scoring system. In an embodiment, theabove-mentioned subject has a NAS of at least 5 as measured by the NASCRN scoring system. In an embodiment, the subject has a NAS of at least6 as measured by the NAS CRN scoring system. In an embodiment, thesubject has a NAS of at least 7 as measured by the NAS CRN scoringsystem. In an embodiment, the subject has a NAS of 8 as measured by theNAS CRN scoring system. The NAS calculated according to the NAS CRNscoring system comprises the sum of grades for steatosis (grades 0-3),hepatocellular ballooning (grades 0-2), and lobular inflammation (grades0-3) (Kleiner D E, et al. Hepatology 2005; 41:1313-21). In anembodiment, the treatment reduces the steatosis score. In an embodiment,the treatment reduces the hepatocellular ballooning score. In anembodiment, the treatment reduces the lobular inflammation score. In anembodiment, the treatment reduces at least two of the steatosis score,the hepatocellular ballooning score and the lobular inflammation score.

In an embodiment, the above-mentioned method or use comprisesidentifying a subject having a NAS of at least 1, 2, 3, 4, 5, 6, 7 or 8as measured by the NAS CRN scoring system.

In another embodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, results in no significant change (i.e. stabilization ornormalization) or a reduction of the NAS over time. In an embodiment,the administration of the GHRH molecule or a pharmaceutically acceptablesalt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ orpharmaceutically acceptable salt thereof, results in a reduction of theNAS score. In an embodiment, the reduction of the NAS score is areduction of at least 0.2. In a further embodiment, the reduction of theNAS score is a reduction of at least 0.3. In a further embodiment, thereduction of the NAS score is a reduction of at least 0.4. In a furtherembodiment, the reduction of the NAS score is a reduction of at least0.5. In a further embodiment, the reduction of the NAS score is areduction of at least 0.6. In a further embodiment, the reduction of theNAS score is a reduction of at least 0.7. In a further embodiment, thereduction of the NAS score is a reduction of at least 0.8. In a furtherembodiment, the reduction of the NAS score is a reduction of at least0.9. In a further embodiment, the reduction of the NAS score is areduction of at least 1.0. In a further embodiment, the reduction of theNAS score is a reduction of at least 2.0.

In an embodiment, the above-mentioned subject has serum ALT levels of atleast about 30 U/L. In an embodiment, the above-mentioned subject hasserum ALT levels of at least about 35 U/L. In another embodiment, theadministration of the GHRH molecule or a pharmaceutically acceptablesalt thereof, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ orpharmaceutically acceptable salt thereof, reduces serum ALT levels by atleast about 10%, 15%, 20%, 25% or 30% in the subject.

In an embodiment, the above-mentioned method or use comprisesidentifying a subject having serum ALT levels of at least about 25, 30U/L or 35 U/L.

In another embodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces serum C-reactive protein (CRP) levels in the subject.

In an embodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces inflammation and/or oxidative stress (e.g., the levelof reactive oxygen (ROS) species) in the liver of the subject. In anembodiment, the reduction of inflammation comprises a reduction of theactivation of inflammatory pathways (e.g., TNF-alpha, IL-6 and/or IL-2pathways) or inflammatory-related genes.

In an embodiment, the administration of the GHRH molecule or apharmaceutically acceptable salt thereof, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof, reduces tissue repair in the liver, e.g., reduces theexpression of genes involved in tissue repair, cell apoptosis, and/orEpithelial to mesenchymal transition (EMT), such as genes from the TGF-βpathway.

In an embodiment, the subject suffers from human immunodeficiency virus(HIV) infection. In an embodiment, the subject is undergoingantiretroviral therapy.

In an embodiment, the treatment or use described herein has no orsubstantially no effect on low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol (HDL-C), or triglycerides in thesubject. In an embodiment, the treatment or use described herein has noor substantially no effect on fasting glucose or hemoglobin A1c in thesubject.

In an embodiment, the subject has a body mass index (BMI) of at leastabout 25. In an embodiment, the subject has a BMI of at least about 26.In an embodiment, the subject has a BMI of at least about 27. In anembodiment, the subject has a BMI of at least about 28. In anembodiment, the subject has a BMI of at least about 29. In anembodiment, the subject has a BMI of at least about 30. In anembodiment, the subject has a BMI of at least about 31. In anembodiment, the subject has a BMI of at least about 32.

In an embodiment, the above-mentioned subject has liver fibrosis. In anembodiment, the above-mentioned method or use comprises identifying asubject having liver fibrosis. In an embodiment, the above-mentionedsubject has stage 1 (in a further embodiment, stage 1A, 1B or 1C) liverfibrosis. In an embodiment, the above-mentioned subject has stage 2liver fibrosis. In an embodiment, the above-mentioned subject has stage3 liver fibrosis. The fibrosis stage is determined using the scoringsystem devised by the Pathology Committee of the NASH Clinical ResearchNetwork (Kleiner D E, et al. Hepatology 2005; 41:1313-21):

Stage 0=no fibrosis;

Stage 1=Perisinusoidal or periportal fibrosis;

-   -   Stage 1A: Mild, zone 3, perisinusoidal;    -   Stage 1B: Moderate, zone 3, perisinusoidal;    -   Stage 1C: Portal/periportal only;

Stage 2=Both perisinusoidal and portal/periportal;

Stage 3=Bridging fibrosis;

Stage 4=Cirrhosis.

In an embodiment, the above-mentioned subject has at least two of thefollowing features (1) to (3): (1) an HFF of at least 15%, preferably ofat least 20%, 25% or 30%; (2) a NAS score of at least 3, preferably ofat least 4, 5 or 6; and (3) liver fibrosis, preferably stage 1C or 2liver fibrosis or worse. In an embodiment, the above-mentioned subjecthas features (1) to (3) noted above. In an embodiment, theabove-mentioned method or use comprises identifying a subject having atleast two, or all of the above-mentioned features (1) to (3).

Preventing progression as used herein in reference to liver fibrosis,refers to no or substantially no progression of liver fibrosis followingtreatment with a GHRH molecule relative to in the absence of suchtreatment. Reducing progression as used herein in reference to liverfibrosis, refers to a situation in which progression of liver fibrosismay continue following treatment with a GHRH molecule, however at areduced rate of progression relative to in the absence of suchtreatment.

In an embodiment, the treatment or use described herein reduces the riskor likelihood that the subject develops liver cancer such ashepatocellular carcinoma (HCC).

The term “GHRH molecule” as used in the context of the presentdisclosure includes, without limitation, human native GHRH₍₁₋₄₄₎ andfragments thereof (e.g., GHRH₍₁₋₄₀₎, GHRH₍₁₋₂₉₎, fragments rangingbetween 1-29 and the 1-44 sequence), and any other fragments; GHRH fromother species and fragments thereof; GHRH variants containing aminoacid(s) substitution(s), addition(s) and/or deletion(s); derivatives oranalogs of GHRH or fragments or variants thereof having for example anorganic group or a moiety coupled to the GHRH amino acid sequence at theN-terminus, the C-terminus or on the side-chain; and pharmaceuticallyacceptable salts of GHRH (human or from other species), as well aspharmaceutically acceptable salts of GHRH fragments, variants, analogsand derivatives. The GHRH molecules of the present disclosure alsoencompass the GHRH molecules currently known in the art, including,without limitation, albumin-conjugated GHRH (U.S. Pat. No. 7,268,113);pegylated GHRH peptide (U.S. Pat. Nos. 7,256,258 and 6,528,485); porcineGHRH (1-40) (U.S. Pat. No. 6,551,996); canine GHRH (U.S. patentapplication no. 2005/0064554); GHRH variants of 1-29 to 1-44 amino acidlength (U.S. Pat. Nos. 5,846,936, 5,696,089, 5,756,458 and 5,416,073,and U.S. patent application Nos. 2006/0128615 and 2004/0192593); andPro⁰-GHRHpeptide and variants thereof (U.S. Pat. No. 5,137,872).

The GHRH analogs include those described in U.S. Pat. Nos. 5,681,379 and5,939,386, which also describe their method of synthesis. Moreparticularly, these GHRH analogs are defined by the following formula A:

X-GHRH Peptide  (A)

wherein the GHRH peptide is a peptide of the following formula B (SEQ IDNO:2):

A1-A2-Asp-Ala-Ile-Phe-Thr-A8-Ser-Tyr-Arg-Lys-A13-Leu-A15-Gln-Leu-A18-Ala-Arg-Lys-Leu-Leu-A24-A25-Ile-A27-A28-Arg-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39-A40-A41-A42-A43-A44-R0  (B)

wherein,

-   -   A1 is Tyr or His;    -   A2 is Val or Ala;    -   A8 is Asn or Ser;    -   A13 is Val or Ile;    -   A15 is Ala or Gly;    -   A18 is Ser or Tyr;    -   A24 is Gln or His;    -   A25 is Asp or Glu;    -   A27 is Met, Ile or Nle    -   A28 is Ser or Asn;    -   A30 is absent or is any amino acid, preferably Gln;    -   A31 is absent or is any amino acid, preferably Gln;    -   A32 is absent or is any amino acid, preferably Gly;    -   A33 is absent or is any amino acid, preferably Glu;    -   A34 is absent or is any amino acid, preferably Ser;    -   A35 is absent or is any amino acid, preferably Asn;    -   A36 is absent or is any amino acid, preferably Gln;    -   A37 is absent or is any amino acid, preferably Glu;    -   A38 is absent or is any amino acid, preferably Arg;    -   A39 is absent or is any amino acid, preferably Gly;    -   A40 is absent or is any amino acid, preferably Ala;    -   A41 is absent or is any amino acid, preferably Arg;    -   A42 is absent or is any amino acid, preferably Ala;    -   A43 is absent or is any amino acid, preferably Arg;    -   A44 is absent or is any amino acid, preferably Leu; and    -   R0 is NH₂ or NH—(CH₂)n-CONH₂, with n=1 to 12.

The group X is a hydrophobic tail anchored via an amide bond to theN-terminus of the peptide and the hydrophobic tail defining a backboneof 5 to 7 atoms. The backbone can be substituted by 01-6 alkyl, 03-6cycloalkyl, or 06-12 aryl and the backbone comprises at least onerigidifying moiety connected to at least two atoms of the backbone. Therigidifying moiety is a double bond, triple bond, saturated orunsaturated 03-9 cycloalkyl, or 06-12 aryl.

In an embodiment, group X is:

In an embodiment, in formula B, A30-A44 are: (a) absent; (b) an aminoacid sequence corresponding to positions 30-44 of a native GHRH peptide(SEQ ID NO: 3), or (c) the amino acid sequence of (b) having a 1-14amino acid deletion from its C-terminus.

In an embodiment, the GHRH peptide is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 4.

In an embodiment, the GHRH molecule is (hexenoyl trans-3)hGHRH₍₁₋₄₄₎NH₂(SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof.[trans-3-hexenoyl]hGHRH₍₁₋₄₄₎ amide (also referred to as tesamorelin and(hexenoyl trans-3)hGHRH₍₁₋₄₄₎NH₂) is a synthetic human GHRH (hGHRH)analog that comprises the 44-amino acid sequence of hGHRH on which ahexenoyl moiety, a C₆ side chain, has been anchored on theamino-terminal tyrosine residue. The structure of[trans-3-hexenoyl]hGHRH₍₁₋₄₄₎ amide is depicted at FIG. 5.

The term “pharmaceutically acceptable salt” refers to a salt of a GHRHmolecule (e.g., trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂) that ispharmacologically acceptable and substantially non-toxic to the subjectto which it is administered. More specifically, these salts retain thebiological effectiveness and properties of the GHRH molecules (e.g.,trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂) and are formed from suitable non-toxicorganic or inorganic acids or bases.

For example, these salts include acid addition salts of GHRH molecules(e.g., trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂) which are sufficiently basic toform such salts. Such acid addition salts include acetates, adipates,alginates, lower alkanesulfonates such as a methanesulfonates,trifluoromethanesulfonatse or ethanesulfonates, arylsulfonates such as abenzenesulfonates, 2-naphthalenesulfonates, or toluenesulfonates (alsoknown as tosylates), ascorbates, aspartates, benzoates,benzenesulfonates, bisulfates, borates, butyrates, citrates,camphorates, camphorsulfonates, cinnamates, cyclopentanepropionates,digluconates, dodecylsulfates, ethanesulfonates, fumarates,glucoheptanoates, glycerophosphates, hemisulfates, heptanoates,hexanoates, hydrochlorides, hydrobromides, hydroiodides, hydrogensulphates, 2-hydroxyethanesulfonates, itaconates, lactates, maleates,mandelates, methanesulfonates, nicotinates, nitrates, oxalates,pamoates, pectinates, perchlorates, persulfates, 3-phenylpropionates,phosphates, picrates, pivalates, propionates, salicylates, succinates,sulfates, sulfonates, tartrates, thiocyanates, undecanoates and thelike.

Additionally, acids which are generally considered suitable for theformation of pharmaceutically useful salts from basic pharmaceuticalcompounds are discussed, for example, by P. Stahl et al., Camille G.(eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use.(2002) Zurich: Wiley-VCH; S. Berge et al, Journal of PharmaceuticalSciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics(1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry(1996), Academic Press, New York; and in The Orange Book (Food & DrugAdministration, Washington, D.C. on their website).

Such salts can be formed quite readily by those skilled in the art usingstandard techniques. Indeed, the chemical modification of apharmaceutical compound (i.e. drug) into a salt is a technique wellknown to pharmaceutical chemists, (See, e.g., H. Ansel et. al.,Pharmaceutical Dosage Forms and Drug Delivery Systems (6^(th) Ed. 1995)at pp. 196 and 1456-1457). Salts of the trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂may be formed, for example, by reacting thetrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ with an amount of acid or base, such asan equivalent amount, in a medium such as one in which the saltprecipitates or in an aqueous medium followed by lyophilization.

In an embodiment, the pharmaceutically acceptable salt of the GHRHmolecule, preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂, is an acetatesalt.

In an embodiment, the GHRH molecule, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂, or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition at a dose of about 1mg/ml to about 10 mg/ml. In a further embodiment, the GHRH molecule,preferably trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂, or pharmaceuticallyacceptable salt thereof is present in a pharmaceutical composition at adose of about 1 mg/ml to about 10 mg/ml, preferably about 1 mg/ml toabout 8 mg/ml or about 4 mg/ml to about 8 mg/ml, for example about 1mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about6 mg/ml, about 7 mg/ml, or about 8 mg/ml.

In an embodiment, the GHRH molecule, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂, or pharmaceutically acceptable saltthereof is present in a pharmaceutical composition comprising one ormore pharmaceutically acceptable excipients.

The term “pharmaceutically acceptable excipient” as used herein has itsnormal meaning in the art and is any ingredient that is not an activeingredient (drug) itself. Excipients include for example binders,lubricants, diluents, bulking agents (fillers), thickening agents,disintegrants, plasticizers, coatings, barrier layer formulations,lubricants, stabilizing agent, release-delaying agents and othercomponents. “Pharmaceutically acceptable excipient” as used hereinrefers to any excipient that does not interfere with effectiveness ofthe biological activity of the active ingredients and that is not toxicto the subject, i.e., is a type of excipient and/or is for use in anamount which is not toxic to the subject. Excipients are well known inthe art, and the present composition is not limited in these respects.In certain embodiments, the pharmaceutical composition comprises one ormore excipients, including for example and without limitation, one ormore binders (binding agents), thickening agents, surfactants, diluents,release-delaying agents, colorants, flavoring agents, fillers,disintegrants/dissolution promoting agents, lubricants, plasticizers,silica flow conditioners, glidants, anti-caking agents, anti-tackingagents, stabilizing agents, anti-static agents, swelling agents and anycombinations thereof. As those of skill would recognize, a singleexcipient can fulfill more than two functions at once, e.g., can act asboth a binding agent and a thickening agent. As those of skill will alsorecognize, these terms are not necessarily mutually exclusive.Therapeutic formulations are prepared using standard methods known inthe art by mixing the active ingredient having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers,excipients and/or stabilizers. The excipient(s) may be suitable, forexample, for intravenous, parenteral, subcutaneous, intramuscular,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraspinal, intrathecal, epidural, intracisternal, intraperitoneal,intranasal or pulmonary (e.g., aerosol) administration (see Remington:The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22^(nd)edition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, byRowe et al., 2012, 7th edition, Pharmaceutical Press). In an embodiment,the pharmaceutical composition is an injectable composition. In anembodiment, the pharmaceutical composition comprises one or moreexcipients for subcutaneous administration/injection.

In an embodiment, the pharmaceutical composition comprises a bulkingagent. The term “bulking agent” as used herein refers to a compound usedto provide an adequate or desired tonicity of the solution resultingfrom the reconstitution of the lyophilized formulation. Preferably, theadequate or desired tonicity of the solution is equal to or approximatesisotonicity with physiological fluid of the subject to which thesolution is administered. For example, one or more sugars may be used asthe bulking agent. Sugars, as used herein, include, but are not limitedto, monosaccharides, oligosaccharides and polysaccharides. Examples ofsuitable sugars include, but are not limited to, mannose, sorbose,xylose, maltose, lactose, sucrose, and dextran. Sugar also includessugar alcohols, such as mannitol, inositol, dulcitol, xylitol andarabitol. Mixtures of sugars may also be used in accordance with thepresent disclosure. In an embodiment, the bulking agent is mannitol. Forexample, one or more amino acids, such as glycine, may be used as thebulking agent. The bulking agent is in concentration of about 1% toabout 10% (w/w) or about 2% to about 8% (w/w) in the pharmaceuticalcomposition. In an embodiment, the bulking agent is in concentration ofabout 3 to about 5% (w/w) in the pharmaceutical composition. In afurther embodiment, the bulking agent is in concentration of about 4%(w/w) in the pharmaceutical composition.

In an embodiment, the pharmaceutical composition of the presentdisclosure may further comprise a surfactant. Typical examples ofsurfactants include sorbitan fatty acid esters such as sorbitanmonocaprylate, sorbitan monolaurate, sorbitan monopalmitate; glycerinfatty acid esters such as glycerin monocaprylate, glycerinmonomyristate, glycerin monostearate; polyglycerin fatty acid esterssuch as decaglyceryl monostearate, decaglyceryl distearate, decaglycerylmonolinoleate; polyoxyethylene sorbitan fatty acid esters such aspolyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monostearate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan trioleate,polyoxyethylene sorbitan tristearate; polyoxyethylene sorbitol fattyacid esters such as polyoxyethylene sorbitol tetrastearate,polyoxyethylene sorbitol tetraoleate; polyoxyethylene glycerin fattyacid esters such as polyoxyethylene glyceryl monostearate; polyethyleneglycol fatty acid esters such as polyethylene glycol distearate;polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether;polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylenepolyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propylether, polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylenealkyl phenyl ethers such as polyoxyethylene nonyl phenyl ether;polyoxyethylene hardened castor oils such as polyoxyethylene castor oil,polyoxyethylene hardened castor oil (polyoxyethylene hydrogenated castoroil); polyoxyethylene beeswax derivatives such as polyoxyethylenesorbitol beeswax; polyoxyethylene lanolin derivatives such aspolyoxyethylene lanolin; polyoxyethylene fatty acid amides such aspolyoxyethylene stearic acid amide; alkyl sulfates having a C₁₀₋₁₈ alkylgroup such as sodium cetyl sulfate, sodium lauryl sulfate, sodium C₁₀₋₁₆sulfate; polyoxyethylene alkyl ether sulfates having an average EO molenumber of 2-4 and a 010-18 alkyl group such as sodium polyoxyethylenelauryl sulfate; alkyl sulfosuccinic acid ester salts having a C₈₋₁₈alkyl group such as sodium laurylsulfosuccinate; lecithin;glycerophospholipids; sphingophospholipids such as sphingomyelin;sucrose fatty acid esters of C₁₂₋₁₈ fatty acids.

In an embodiment, the surfactant is a non-ionic surfactant. In a furtherembodiment, the surfactant is polyoxyethylene sorbitan alkyl ester. Inyet a further embodiment, the surfactant is polysorbate-20 (T20 orTween-20™).

In an embodiment, the pharmaceutical composition of the presentdisclosure may further comprise one or more stabilizing agents orstabilizers. As used herein, the term “stabilizer” is intended to mean acompound used to stabilize the therapeutic agent against physical,chemical, or biochemical process that would reduce the therapeuticactivity of the agent. Suitable stabilizers are non-reducing sugarsincluding, by way of example and without limitation, sucrose (orsaccharose) and trehalose; and non-reducing polyols including, by way ofexample and without limitation, sorbitol, mannitol, maltitol, xylitol,glycol, glycerol and ethylene glycol. In an embodiment, thepharmaceutical composition comprises about 2% to about 10% (w/v) ofmannitol. In a further embodiment, the pharmaceutical compositioncomprises about 2% to about 8% (w/v), about 3% to about 7% (w/v), about4% to about 6% (w/v), or about 5% (w/v), of mannitol.

In an embodiment, the pharmaceutical composition of the presentdisclosure comprises a non-reducing sugar. “Non-reducing sugar” as usedherein refers to a sugar that does not contain a hemi-acetal, forexample a carbohydrate or sugar characterized by having a glycosidicbond formed between the reducing ends of the sugar units, and notbetween a reducing end of one sugar unit and a non-reducing end of theother sugar unit. In a further embodiment, the above-mentionednon-reducing sugar is trehalose or sucrose. In a further embodiment, theabove-mentioned non-reducing sugar is sucrose. In an embodiment, thenon-reducing sugar is in a concentration of about 0.1% to about 5% (w/w)in the pharmaceutical composition of the disclosure. In an embodiment,the non-reducing sugar is in a concentration of about 1% to about 3%(w/w). In a further embodiment, the non-reducing sugar is in aconcentration of about 2% (w/w).

In an embodiment, the pharmaceutical composition of the presentdisclosure comprises a buffering agent, i.e. an agent that maintains thepH of the pharmaceutical composition near a chosen value. Examples ofbuffering agents include acetate buffers, succinate buffers, citratebuffers, phosphate buffers and histidine buffers. In an embodiment, thebuffering agent is a histidine buffer. In an embodiment, theconcentration of histidine in the pharmaceutical composition is about0.01% to about 1%, for example about 0.05% to about 0.5% or about 0.1%to about 0.3%. In a further embodiment, the histidine sugar is in aconcentration of about 0.15%.

In another embodiment, the amount of surfactant in the pharmaceuticalcomposition of the present disclosure is about 0.0001% to about 10%(w/w). In a further embodiment, the amount of surfactant in thepharmaceutical composition of the present disclosure is about 0.001% toabout 5%, 1% or 0.1% (w/w) or about 0.005% to about 0.05%. In yet afurther embodiment, the amount of surfactant in the pharmaceuticalcomposition of the present disclosure is about 0.01% (w/w).

In an embodiment, the pharmaceutical composition of the presentdisclosure comprises an oligosaccharide, for example a cyclicoligosaccharide such as a cyclodextrin. The term “cyclodextrin” as usedherein refers to a family of cyclic oligosaccharides, comprising amacrocyclic ring of glucopyranoside subunits (5 or more) joined by α-1,4glycosidic bonds. Examples of cyclodextrins include α-cyclodextrin,β-cyclodextrin and γ-cyclodextrin, which comprise 6, 7 and 8glucopyranoside subunits, respectively, as well as analogs thereof(e.g., modified cyclodextrins). In an embodiment, the cyclodextrin is aβ-cyclodextrin or a modified β-cyclodextrin. A modified β-cyclodextrinas used herein refers to a β-cyclodextrin molecule in which one or moreof the hydroxyl groups of one or more of the sugar units may bemodified, for example with an alkyl, alkenyl or alkynyl group, or with asubstituted alkyl, alkenyl or alkynyl group. Therefore, in embodiments,the β-cyclodextrin may be unmodified or unsubstituted, or may bemodified or substituted. As such, in a further embodiment, theβ-cyclodextrin is a modified β-cyclodextrin. “Modified β-cyclodextrin”as used herein refers to a β-cyclodextrin that contains a modificationat one or more hydroxyl groups of one or more sugar units of theβ-cyclodextrin, i.e., a group or moiety that is attached to one or morehydroxyl groups of one or more sugar units of the β-cyclodextrin. Assuch, in embodiments, the modified β-cyclodextrin is an alkyl-,alkenyl-, alkynyl, substituted alkyl-, substituted alkenyl orsubstituted alkynyl-β-cyclodextrin (e.g., with a hydroxyl substitution).In embodiments, the alkyl, alkenyl or alkynyl groups are (C₁-C₆)alkyl,(C₁-C₆)alkenyl or (C₁-C₆)alkynyl groups. In a further embodiment, themodified β-cyclodextrin is a (C₁-C₆)alkyl β-cyclodextrin, in a furtherembodiment methyl-β-cyclodextrin (M-R-CD). In a further embodiment, themodified β-cyclodextrin is a hydroxy(C₁-C₆)alkyl pcyclodextrin, in afurther embodiment hydroxypropyl-β-cyclodextrin (HP-β-CD). In anembodiment, the cyclodextrin is present in the pharmaceuticalcomposition at a concentration of about 2 to about 15% (w/v), in afurther embodiment about 2 to about 12.5% (w/v), for example about 2 toabout 10% (w/v), about 2.5 to about 15% (w/v), about 2.5 to about 12.5%(w/v), about 2.5 to about 10% (w/v), about 5 to about 15% (w/v), about 5to about 12.5% (w/v), about 5 to about 10% (w/v), about 7.5 to about12.5% (w/v), about 7.5 to about 10% (w/v), about 5, 7.5, 10, 12.5 or 15%(w/v), or about 10% (w/v).

In an embodiment, the pharmaceutical composition of the presentdisclosure has a pH of about 4.5 to about 6.5, for example about 5.0 toabout 6.0. According to another embodiment, the pharmaceuticalcomposition has a pH of about 5.0. According to a further embodiment,the pharmaceutical composition has a pH of about 5.5. According toanother further embodiment, the pharmaceutical composition has a pH ofabout 6.0.

In an embodiment, the pharmaceutical composition of the presentdisclosure comprises a diluent, for example an aqueous solution. In afurther embodiment, the pharmaceutical composition comprises (typicallysterile) water.

The pharmaceutical composition of the present disclosure may furthercontain other diluents, solubilizing agents, excipients, pH-modifiers,soothing agents, buffers, sulfur-containing reducing agents,antioxidants or the like, if desired. For example, sulfur-containingreducing agents include N-acetylcysteine, N-acetylhomocysteine, thiocticacid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol,thioglycolic acid and salts thereof, sodium thiosulfate, glutathione,methionine and sulfhydryl-containing compounds such as thioalkanoic acidhaving 1 to 7 carbon atoms. Antioxidants include methionine, erythorbicacid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol,tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbylpalmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite,triamyl gallate, propyl gallate or chelating agents such as disodiumethylenediamine tetraacetate (EDTA), sodium pyrophosphate, sodiummetaphosphate. Other components commonly added may also be contained,e.g., inorganic salts such as sodium chloride, potassium chloride,calcium chloride, sodium phosphate, potassium phosphate, sodiumbicarbonate; and organic salts such as sodium citrate, potassiumcitrate, sodium acetate.

In an embodiment, the GHRH molecule, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂, or pharmaceutically acceptable saltthereof is administered at a daily dose of about 1 mg to about 8 mg. Ina further embodiment, the GHRH molecule, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂, or pharmaceutically acceptable saltthereof is administered at a daily dose of about 1 mg to about 4 mg,about 1 mg to about 3 mg or about 1 mg to about 2 mg. In a furtherembodiment, the GHRH molecule, preferablytrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂, or pharmaceutically acceptable saltthereof is administered ata daily dose of about 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mg.

In other aspects, the present disclosure provides a use of theabove-mentioned GHRH molecule or composition for achieving one or moreof the biological/therapeutic effects noted herein, e.g., for improving,reducing the progression of, stabilizing, reducing the severity of,preventing and/or treating the conditions, diseases or disorders notedherein, or for the preparation/manufacture of a medicament forimproving, reducing the progression of, stabilizing, reducing theseverity of, preventing and/or treating the conditions, diseases ordisorders noted herein. In other aspects, the present disclosureprovides the above-mentioned composition for use in improving, reducingthe progression of, stabilizing, reducing the severity of, preventingand/or treating the conditions, diseases or disorders noted herein, orfor the preparation/manufacture of a medicament for improving, reducingthe progression of, reducing the severity of, preventing and/or treatingthe conditions, diseases or disorders noted herein.

The term “treatment” as used herein, is defined as the application oradministration of a therapeutic agent to a subject, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a subject, who has a disorder, a disease, a symptom of disorder ordisease, or a predisposition toward a disorder or disease, with thepurpose to cure, heal, alleviate, delay, relieve, alter, remedy,ameliorate, improve or affect the disorder/disease, the symptoms ofdisorder/disease or the predisposition toward disorder/disease.

In an embodiment, the treatment is for a period of at least 3, 6, 9 or12 months. In a further embodiment, the treatment is for a period of atleast 12 months.

In embodiments, the GHRH molecule is for administration in an effectiveamount, e.g., a therapeutically effective amount or a prophylacticallyeffective amount. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result, such as to effect theabove-noted alterations and to reduce the progression of the above-notedconditions. A therapeutically effective amount of a GHRH molecule mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the compound to elicit adesired response in the individual. Dosage regimens may be adjusted toprovide the optimum therapeutic response. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of thecompound are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result, such as preventing or inhibiting the rate of onsetor progression of the above-noted conditions. A prophylacticallyeffective amount can be determined as described above for thetherapeutically effective amount. For any particular subject, specificdosage regimens may be adjusted over time according to the individualneed and the professional judgement of the person administering orsupervising the administration of the compositions.

In accordance with another aspect of the disclosure, therapeuticcompositions of the present disclosure, comprising a GH secretagogue,may be provided in containers, kits or packages (e.g. commercialpackages) which further comprise instructions for its use to prevent ortreat the conditions noted herein.

Accordingly, the disclosure further provides a kit or package comprisinga GHRH molecule or the above-mentioned composition, optionally togetherwith instructions to prevent or treat the conditions noted herein.

As used herein, the term “subject” or “patient” are taken to mean awarm-blooded animal such as a mammal, for example, a cat, a dog, amouse, a guinea pig, a horse, a bovine cow, a sheep or a human. In anembodiment, the subject is a mammal. In a further embodiment, thesubject is a human.

EXAMPLES

The present disclosure is illustrated in further detail by the followingnon-limiting examples.

Example 1: Materials and Methods

Participant Selection

Participants were recruited at the Massachusetts General Hospital (MGH,Boston, Mass.) and the National Institutes of Health (NIH, Bethesda,Md.). Potentially eligible participants were identified throughreferrals from local physicians, notices sent to participants enrolledin the Research Subject Volunteer Program at MGH, and the NIH cohort ofpatients with HIV and NAFLD. Sixty-one men and women with HIV-infectionmet eligibility criteria and participated in a baseline assessment.

Participants were eligible for the study if they were between 18-70years of age and had confirmed HIV infection as well as hepaticsteatosis as demonstrated by liver fat fraction of ≥5% on magneticresonance spectroscopy (MRS). Participants with heavy alcohol use (>20 gdaily for women or >30 g daily for men) were excluded, as wereparticipants with hepatitis B, active hepatitis C, alpha-1 antitrypsindeficiency, Wilson's disease, hemochromatosis, or autoimmune hepatitis.Those with a history of hepatitis C were only eligible if they had beensuccessfully treated, with resolution of hepatitis C at least one yearprior to study entry and an undetectable HCV viral load at the screeningvisit. Participants with known cirrhosis, Stage 4 fibrosis on biopsy, orother severe chronic illness were also excluded. Participants with milddiabetes were eligible as long as hemoglobin A1c ≤7%, anti-diabeticagents were stable for months, and they were not using insulin orthiazoledinediones. Participants were also required to have a stableantiviral regimen for months, stable use of any antihypertensive orlipid lowering medications for months, and, if applicable, stable use ofvitamin E for ≥6 months prior to study entry. Participants using chronicsystemic corticosteroids, methotrexate, amiodarone, tamoxifen, or GHwere excluded, as were participants with any active malignancy. Women50y or older were required to have a negative mammogram within 1 year ofthe baseline visit, and men with history of prostate cancer wereexcluded. Participants with history of hypopituitarism or otherconditions known to affect the GH axis were also ineligible. Otherexclusionary labs for safety reasons were as follows: hemoglobin <11g/dL, CD4⁺ count <100 cells/mm³, HIV viral load >400 copies/mL, andprostate specific antigen <5 ng/mL.

Study Design

The study consisted of a 12-month, double-blind treatment phase duringwhich participants were randomized in a 1:1 ratio receive to tesamorelin2 mg daily or identical placebo, followed by a 6-month open label phaseduring which all participants receive tesamorelin. The pre-specifiedprimary analysis, comparing changes over twelve months in tesamorelinvs. placebo treatment, is reported here. Randomization was stratified bysite (NIH and MGH) and vitamin E use, defined as consistent use of 400international units daily. The randomization list was prepared by thestudy statistician using a permuted block algorithm within each stratumwith randomly varying block sizes. Tesamorelin was administered at theFDA-approved dose of 2 mg subcutaneously daily; participants weretrained in reconstitution and self-injection at the baseline visit andadministered the injections at home, returning used vials to assesscompliance. IGF-1 Z-scores were monitored throughout the study by anindependent endocrinologist at MGH otherwise unaffiliated with thestudy. A pre-specified threshold of IGF-1 Z-score of was included in theprotocol as a trigger to decrease tesamorelin dose to 1 mg, along with adummy dose-reduction in the placebo group, but this was not required forany patient during the double-blind phase of the study.

All subjects received nutritional counseling from clinical researchnutritionists at baseline, six and 12 months. Visits were conducted inthe fasting state. The screening visit included history and physicalexamination, laboratory investigations for eligibility, MRS and magneticresonance imaging (MRI), with the latter used to assess cross-sectionalarea of visceral adipose tissue (VAT) at the L4 vertebra. The MRI/MRS,HbA1c, CD4⁺ count, and HIV viral load from the screening visit were usedas the baseline measures. The baseline assessment included liver biopsy;whole body dual energy x-ray absorptiometry (DXA); fasting assessment ofliver function tests, lipids, serum inflammatory markers, IGF-1, andHbA1c; and bionutrition assessment including 4-day food record,Modifiable Activity Questionnaire (Kriska A M, et al. Diabetes Care1990; 13:401-11), and anthropometric measures performed in triplicate.Baseline assessments were repeated at 12 months along with repeatMRI/MRS, HbA1c, and immunologic parameters. An interim MRI/MRS wasperformed at the 6-month visit.

Outcome Measures

The primary outcome was HFF as measured using ¹H MRS, performed in themorning following an 8-hour fast. Fat fraction was calculated as thearea under the spectroscopic lipid peak divided by the total area underthe water and lipid peaks. Image acquisition followed a standardprotocol at MGH and NIH. Liver fat content was quantified using jMRUIsemi-automated software at NIH and automated LC-Model software at MGH.NIH scans were re-read at MGH using LC-Model, and the correlationbetween measurements was 0.97, equivalent to the correlation between twoscans performed on the same machine before and after repositioning andanalyzed using LC-Model (Bredella M A, et al. J Comput Assist Tomogr2010; 34:372-6). The diagnostic accuracy of MRS for liver steatosis hasan area under the receiver operating characteristic curve of 0.94 (95%CI 0.88-1.0) compared to assessment of liver biopsy by an experiencedpathologist (Georgoff P, et al. AJR Am J Roentgenol 2012; 199:2-7).Subjects were eligible if HFF was 5%.

Histological scoring was performed by a blinded central pathologist(DEK) for all liver biopsy samples using the NAS CRN scoring system(Kleiner D E, et al. Hepatology 2005; 41:1313-21). The sum of grades forsteatosis (grades 0-3), hepatocellular ballooning (grades 0-2), andlobular inflammation (0-3) comprise the NAS, and fibrosis isindependently staged between 0-4 (Kleiner D E, et al. Hepatology 2005;41:1313-21). Presence or absence of steatohepatitis was determined byhistological review (DEK). Progression of fibrosis was considered anyincrease in fibrosis stage between baseline and 12 months.Cross-sectional MRI of the abdomen at the L4 vertebra was read centrallyand used to quantify VAT and subcutaneous adipose tissue (SAT) areas.

Participants at MGH also underwent a euglycemic hyperinsulinemic clampprocedure to assess insulin sensitivity. After a 14-hour overnight fast,a low-dose (insulin 20 mU/m²/min) clamp for 2 hours was followed by ahigh-dose (insulin 80 mU/m²/min) clamp for 2 hours as previouslydescribed (Braun L R, et al. Effects of Pitavastatin on InsulinSensitivity and Liver Fat: A Randomized Clinical Trial. J ClinEndocrinol Metab 2018; 103:4176-86). Insulin stimulated glucose disposal(M) were calculated during the last 20 minutes of low-dose and high-doseclamp using the DeFronzo method (DeFronzo R A, Tobin J D, Andres R. Am JPhysiol 1979; 237:E214-23) as the primary indices of hepatic andwhole-body insulin sensitivity, respectively.

Laboratory analyses were conducted using standard methodologies.Clinical labs were measured at the NIH Clinical Laboratory and, for MGH,at LabCorp and Quest. IGF-1 was measured centrally at QuestLaboratories. C-reactive protein was measured usingelectrochemiluminescense (Meso Scale Discovery, Rockville, Md.), andadiponectin was measured using ELISA (R&D Systems, Minneapolis, Minn.).

Statistical Analysis

The pre-specified primary endpoint was change in HFF between baselineand 12 months. Secondary endpoints included change in liver histology,ALT, lipids, measures of glucose metabolism, and markers of systemicinflammation. A sample size of 60 was chosen based on 80% power todetect a treatment difference of 0.85 standard deviation change inhepatic fat fraction over 12 months, assuming a discontinuation rate of25%, i.e., 45 evaluable patients, at a two-sided alpha of 0.05. Afterone patient at the MGH site discontinued at the conclusion of thebaseline visit, IRB permission was obtained to enroll a 61stparticipant.

Per the pre-specified analysis plan, change in HFF was assessed byrandom intercept mixed effects modeling using restricted maximumlikelihood to assess the effect estimate for the timexrandomizationinteraction. All available data were used in the analysis, which wasbased on intention to treat. The same analysis was used for otherendpoints measured at multiple timepoints during the double-blindperiod, including VAT, SAT, ALT, BMI, and glucose. For secondaryendpoints measured at only baseline and 12-months, a paired t-test wasperformed using all available data. Data are presented as mean±standarddeviation or, for categorical variables, number and percent. Betweengroup comparisons at baseline were assessed using Student's t-test forcontinuous variables and Pearson's Chi Square statistic for categoricalvariables. Pearson's correlation coefficient was used to assessrelationships between continuous variables. Two data points one baselineALT value and one baseline CRP value were excluded due to being morethan 5 standard deviations above the sample mean. A two-sided alpha of0.05 was the pre-defined threshold for statistical significance. Studydata were collected and managed using Research Electronic Data Capture(REDCap) tools hosted at Partners HealthCare (Harris P A, et al. JBiomed Inform 2009; 42:377-81). Sensitivity analyses were performedutilizing multiple imputation of missing data with 100 iterations,discarding the first 10. All data analysis was overseen by the studystatistician (HL).

Example 2: Results

Patients' Characteristics, Adherence

Of 143 total participants screened, 61 entered the randomized treatmentportion of the trial. Fifteen participants were recruited at NIH and 46at MGH. Participant flow and reasons for patient exclusion are shown inFIG. 1. Four participants in the tesamorelin group and two in theplacebo group discontinued before any follow-up imaging, as shown inFIG. 1. Five participants in the tesamorelin group and two in theplacebo group discontinued after obtaining follow-up imaging that wasused in the primary analysis. The overall discontinuation rate was notsignificantly different between groups, P=0.12.

Clinical characteristics (Table 1) and measures of body composition andmetabolism (FIG. 2) were similar between groups at baseline (FIG. 2). Atbaseline, 33% of the cohort had a histologic diagnosis of NASH.Forty-three percent of the cohort had fibrosis Stage 1 or higher; perprotocol, none had Stage 4 fibrosis at baseline. These rates weresimilar in the treatment groups (Table 1). ART regimen was similarbetween groups (Table 1).

TABLE 1 Baseline Demographics and Clinical Characteristics TesamorelinPlacebo (n = 31) (n = 30) Sex (N [%] Male) 24 [77.4%] 24 [80.0%] Age (y)52 ± 8 54 ± 7 51 [47, 56] 55 [49, 60] Race (N [%]) White 21 [67.7%] 19[63.3%] Black 8 [25.8%] 10 [33.3%] Other 2 [6.5%] 1 [3.3%] Ethnicity (N[%] Hispanic) 6 [19.4%] 3 [10.0%] Smoking Status (N [%]) Ever Smoker 15[53.6%] 12 [52.2%] Current Smoker* 4 [12.9%] 7 [23.3%] Alcohol Use(drinks/week)  0.3 ± 1.3  0.9 ± 2.0 Duration of HIV Infection 16 ± 9 18± 8 16 [10, 22] 20 [11, 24] Current Antiretroviral Use (N [%]) NRTI 27[87.1%] 29 [96.7%] PI 9 [29.0%] 6 [20.0%] NNRTI 12 [38.7%] 11 [36.7%]Integrase Inhibitors 21 [67.7%] 18 [60%] Entry Inhibitor 1 [3.2%] 0 [0%]Type 2 Diabetes (N [%]) 4 [12.9%] 4 [13.3%] Current use ofanti-diabetics 3 [9.7%] 3 [10.0%] Current metformin use 3 [9.7%] 2[6.7%] Current Lipid Lowering Medications (N [%]) 13 [41.9%] 15 [50.0%]Current statin use 10 [32.3%] 14 [46.7%] Current Vitamin E Use (N [%]) 2[6.5%] 1 [3.3%] Hepatic Fat (%) 14.7 ± 9.0 12.9 ± 7.7 NASH (N [%]) 10[34.5%] 9 [31.0%] Fibrosis (N [%]) 14 [48.3%] 11 [37.9%] There were nostatistically significant differences between groups at baseline for anyof the variables shown above. Continuous variables are presented as mean± standard deviation. *Current smokers are also represented in the “EverSmoker” category. **Vitamin E use defined as regular use of ≥400international units daily. Abbreviations: g, grams; HIV, humanimmunodeficiency virus; kcal, kilocalories; NASH, nonalcoholicsteatohepatitis, NRTI, nucleoside reverse transcriptase inhibitor; PI,protease inhibitor; NNRTI nonnucleoside reverse transcriptase inhibitor.

Adherence to daily injections by count of returned empty vials wassimilar between treatment groups: 87±16% for placebo and 80±15% fortesamorelin (P=0.11). Change in IGF-1 values, shown in FIG. 2,demonstrate the expected effect of tesamorelin to increase IGF-1, withan effect size of 117 ng/mL (95% CI [76, 157], P<0.0001). No subjectshad IGF-1 Z-scores over the pre-specified dose-reduction threshold(Z-score >3), but one subject received a dose reduction to 1 mg forsymptoms potentially related to growth hormone. This subjectself-discontinued from the study soon after the dose reduction.

Hepatic Fat Fraction, Liver Histology, and Markers of Inflammation

Table 2A shows the hepatic fat fraction (HFF) at baseline and at the endof the 1-year treatment in tesamorelin- and placebo-treated subjects whocompleted the study. Table 2B shows the NASH score, steatosis score,hepatocellular ballooning score and lobular inflammation score atbaseline and at the end of the 1-year treatment in tesamorelin- andplacebo-treated subjects who completed the study. Table 2C shows thefibrosis stage at baseline and at the end of the 1-year treatment intesamorelin- and placebo-treated subjects who completed the study.

TABLE 2A Hepatic Fat Fraction (HFF) Patient Change Change No. TreatmentBaseline Year 1 (absolute) (relative) 2 Tesamorelin 8.10 2.94 −5.16 −64%3 Tesamorelin 11.88 3.85 −8.03 −68% 4 Tesamorelin 10.60 7.87 −2.73 −26%7 Tesamorelin 16.97 2.87 −14.1 −83% 12 Tesamorelin 5.38 1.78 −3.6 −67%13 Tesamorelin 23.96 23.08 −0.88  −4% 18 Tesamorelin 5.36 1.59 −3.77−70% 20 Tesamorelin 9.09 10.22 1.13  12% 24 Tesamorelin 33.19 17.40−15.79 −48% 26 Tesamorelin 15.03 2.14 −12.89 −86% 28 Tesamorelin 14.2610.22 −4.04 −28% 29 Tesamorelin 19.91 19.89 −0.02  0% 31 Tesamorelin11.67 5.97 −5.7 −49% 34 Tesamorelin 7.50 5.42 −2.1 −28% 36 Tesamorelin9.10 23.93 14.83 163% 37 Tesamorelin 13.02 8.86 −4.16 −32% 50Tesamorelin 24 20 −4 −17% 51 Tesamorelin 5 1.4 −3.6 −72% 53 Tesamorelin17 9 −8 −47% 55 Tesamorelin 33.33 21 −12.33 −37% Average Tesamorelin14.72 9.97 −4.75 −32% Median Tesamorelin 12.75 8.37 −4.02 −42% 5 Placebo5.27 8.72 3.45  65% 6 Placebo 11.54 7.73 −3.81 −33% 8 Placebo 6.85 9.572.72  40% 10 Placebo 45.38 39.18 −6.2 −14% 11 Placebo 28.45 24.43 −4.02−14% 15 Placebo 4.98 1.09 −3.89 −78% 16 Placebo 14.27 21.68 7.41  52% 17Placebo 11.64 12.63 0.99  9% 19 Placebo 10.00 10.13 0.13  1% 21 Placebo6.24 14.14 7.9 127% 23 Placebo 18.97 25.46 6.49  34% 27 Placebo 5.505.09 −0.41  −7% 30 Placebo 11.43 8.90 −2.53 −22% 32 Placebo 13.94 12.37−1.57 −11% 33 Placebo 25.94 20.51 −5.43 −21% 35 Placebo 7.42 6.04 −1.38−19% 38 Placebo 17.85 21.65 3.8  21% 39 Placebo 11.73 13.20 1.47  13% 42Placebo 5.90 9.29 3.39  57% 44 Placebo 13.27 15.44 2.17  16% 47 Placebo16.6 16.1 −0.5  −3% 48 Placebo 30 33 3  10% 52 Placebo 16 11 −5 −31% 54Placebo 14.63 10.3 −4.6 −31% 60 Placebo 10 5.55 −4.45 −45% AveragePlacebo 14.55 14.53 −0.035  5% Median Placebo 11.73 12.37 −0.41  −3%

TABLE 2B Lobular Patient NASH score Steatosis inflammation BallooningNo. Treatment Base Year 1 Base Year 1 Base Year 1 Base Year 1 2 Tesa 2 11 1 1 0 0 0 3 Tesa 3 4 1 1 1 2 1 1 4 Tesa 2 2 1 1 1 1 0 0 12 Tesa 2 1 10 1 1 0 0 13 Tesa 3 1 2 1 1 0 0 0 18 Tesa 1 1 0 0 1 1 0 0 20 Tesa 3 2 11 1 1 1 0 24 Tesa 3 3 2 2 1 1 0 0 28 Tesa 2 1 1 1 1 0 0 0 29 Tesa 2 4 12 1 1 0 1 31 Tesa 2 2 1 1 1 1 0 0 34 Tesa 1 1 0 0 1 1 0 0 36 Tesa 1 4 13 0 1 0 0 37 Tesa 0 1 0 1 0 0 0 0 50 Tesa 7 7 2 3 3 2 2 2 51 Tesa 2 1 10 1 1 0 0 53 Tesa 3 3 1 1 1 1 1 1 55 Tesa 7 4 2 2 3 1 2 1 5 Placebo 2 21 1 1 1 0 0 6 Placebo 2 2 1 1 1 1 0 0 8 Placebo 3 2 1 1 2 1 0 0 10Placebo 7 7 3 3 2 2 2 2 11 Placebo 3 4 2 2 1 1 0 1 15 Placebo 1 1 0 0 11 0 0 16 Placebo 2 2 1 1 1 1 0 0 17 Placebo 2 2 1 1 1 1 0 0 19 Placebo 42 1 1 1 1 2 0 21 Placebo 4 5 3 2 1 2 0 1 23 Placebo 3 2 2 1 1 1 0 0 27Placebo 2 1 1 0 1 1 0 0 30 Placebo 2 2 1 1 1 1 0 0 32 Placebo 1 2 0 1 11 0 0 33 Placebo 4 3 2 2 1 1 1 0 35 Placebo 1 1 0 1 1 0 0 0 38 Placebo 47 1 2 2 3 1 2 42 Placebo 1 2 0 1 1 1 0 0 44 Placebo 2 3 1 1 1 2 0 0 47Placebo 7 6 3 3 2 1 2 2 48 Placebo 5 6 3 3 1 2 1 1 52 Placebo 2 3 1 1 11 0 1 56 Placebo 2 2 1 1 1 1 0 0 60 Placebo 2 2 1 1 1 1 0 0

TABLE 2C Patient Fibrosis stage No. Treatment Baseline Year 1 2Tesamorelin 0 0 3 Tesamorelin 3 4 4 Tesamorelin 0 0 12 Tesamorelin 0 013 Tesamorelin 0 0 18 Tesamorelin   1B 0 20 Tesamorelin   1A   1A 24Tesamorelin 0 0 28 Tesamorelin 0 0 29 Tesamorelin   1C   1C 31Tesamorelin 0 0 34 Tesamorelin 2 2 36 Tesamorelin 0 0 37 Tesamorelin 0 050 Tesamorelin 3 3 51 Tesamorelin 0 0 53 Tesamorelin 2 3 55 Tesamorelin2   1A 5 Placebo   1C 0 6 Placebo 0 0 8 Placebo 3   1C 10 Placebo 2 2 11Placebo 0 2 15 Placebo 0 0 16 Placebo 0 0 17 Placebo 0 0 19 Placebo 2  1A 21 Placebo 2 3 23 Placebo 0 0 27 Placebo 0 0 30 Placebo 0   1A 32Placebo   1A   1B 33 Placebo 0 0 35 Placebo 0 0 38 Placebo 2 3 42Placebo 0   1C 44 Placebo 0   1C 47 Placebo 3 3 48 Placebo   1B 2 52Placebo 0   1B 56 Placebo 0 0 60 Placebo 0 0

Tesamorelin significantly reduced HFF compared to placebo (effect size−4.1% (95% CI −7.5, −0.7), P=0.02) compared to placebo. The changebetween baseline and 12 months, shown in FIG. 3A, corresponded to a −37%(95% CI −67, −7) relative change in liver fat (FIG. 3B). In thetesamorelin group, 35% of individuals had a reduction in HFF to <5%,whereas this occurred in 4% of individuals in the placebo group (FIG.3C, P=0.007 for comparison). Also, tesamorelin was effective at reducingHFF in the most afflicted subjects (i.e. subjects having the mostelevated baseline HFF, e.g., 15%), with some patients showing absolutereduction of HFF of more than 10%: from 16.97 to 2.87 for patient no. 7;from 33.19 to 17.4 for patient no. 24; from 15.03 to 2.14 for patientno. 26; and from 33.3 to 21 for patient no. 55 (Table 2A).

Among the entire cohort, tesamorelin prevented the progression offibrosis during the treatment period, with 2 individuals showingprogression in the tesamorelin group versus 9 in the placebo group(P=0.04) (FIG. 4A). Changes in fibrosis during the study were positivelyassociated with changes in NAS score (p-value for ANOVA=0.0003, FIG.4B). Among the entire cohort, tesamorelin did not significantly changeNAS score relative to placebo (effect size −0.3, 95% CI −1.0, 0.5).However, among individuals randomized to tesamorelin, those with higherbaseline NAS scores had greater reductions in their scores duringtreatment (r=−0.48, P=0.04), whereas a similar relationship was notobserved in the placebo group (r=−0.14, P=0.52). Notably, the NAS scoreof patient No. 55 treated with tesamorelin was reduced from 7 to 4,which was associated with an improvement of fibrosis (from stage 2 to1A) (Tables 2B and 2C). Similarly, in the tesamorelin-treated cohort,changes in NAS score during the treatment period were positivelyassociated with changes in HFF (r=0.51, P=0.03), whereas a similarrelationship was not seen in placebo-treated patients (r=0.14, P=0.52).

Relative to placebo, tesamorelin did not significantly reduce ALT or GGTover the treatment period, although both effect sizes suggest a modestreduction (FIG. 2). Restricting the cohort to those with elevated ALT(>30 U/L) at baseline, tesamorelin did significantly decrease ALT after12 months (effect size −29 U/L [95% CI −3, −55], P=0.03, FIG. 3D)relative to placebo. Tesamorelin reduced CRP (FIG. 2) relative toplacebo but did not have any effect on adiponectin.

Lipid and Glucose Metabolism and Body Composition

No effect of tesamorelin on low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol (HDL-C), or triglycerides (FIG. 2)was seen in the present study. Tesamorelin did not significantly affectfasting glucose or hemoglobin A1c during the treatment period (FIG. 2).In the subset of patients who underwent euglycemic hyperinsulinemicclamp, tesamorelin did not affect the glucose infusion rate requiredduring the low-dose clamp (treatment effect for low-dose M 0.0 mg/kg/min[95% CI −1.1, 1.1], P=0.996) nor the insulin stimulated glucose uptakeduring the high-dose clamp (treatment effect for high-dose M −0.9mg/kg/min [95% CI −2.4, 0.7].

Table 3 shows the visceral adiposity at baseline and at the end of the1-year treatment in tesamorelin- and placebo-treated subjects whocompleted the study.

TABLE 3 Visceral adiposity (cm²) Relative Patient No. Treatment BaselineYear 1 change 2 Tesamorelin 130.79 107.84 −18% 3 Tesamorelin 218.12242.96  11% 4 Tesamorelin 96.30 80.86 −16% 7 Tesamorelin 171.49 146.32−15% 12 Tesamorelin 176.60 174.63  −1% 13 Tesamorelin 250.91 244.77  −2%18 Tesamorelin 151.59 141.88  −6% 20 Tesamorelin 159.08 109.01 −31% 24Tesamorelin 238.57 240.79  1% 26 Tesamorelin 203.14 28.55 −86% 28Tesamorelin 332.87 346.80  4% 29 Tesamorelin 329.31 424.63  29% 31Tesamorelin 191.63 98.62 −49% 34 Tesamorelin 251.99 250.21  −1% 36Tesamorelin 329.89 290.85 −12% 37 Tesamorelin 131.36 322.94 146% 50Tesamorelin 300.71 240.42 −20% 51 Tesamorelin 265.39 133.00 −50% 53Tesamorelin 446.64 352.00 −21% 55 Tesamorelin 364.66 335.48  −8% AverageTesamorelin 237.05 215.63  −7% Median Tesamorelin 228.35 221.02 −10% 5Placebo 248.72 338.55  36% 6 Placebo 122.45 132.92  9% 8 Placebo 147.46181.43  23% 10 Placebo 394.51 346.46 −12% 11 Placebo 252.69 257.89  2%15 Placebo 136.05 176.25  30% 16 Placebo 311.93 335.45  8% 17 Placebo220.00 225.39  2% 19 Placebo 286.46 272.48  −5% 21 Placebo 166.76 169.40 2% 23 Placebo 193.37 233.44  21% 27 Placebo 86.57 94.40  9% 30 Placebo245.57 317.52  29% 32 Placebo 339.45 402.50  19% 33 Placebo 291.68272.24  −7% 35 Placebo 116.14 134.17  16% 38 Placebo 230.47 141.44 −39%39 Placebo 243.94 304.42  25% 42 Placebo 190.16 231.52  22% 44 Placebo493.68 562.60  14% 47 Placebo 259.48 238.15  −8% 48 Placebo 476.77448.03  −6% 52 Placebo 357.75 356.78  0% 54 Placebo 138.49 141.17  2% 56Placebo 235.52 241.11  2% 60 Placebo 122.65 118.99  −3% Average Placebo242.64 256.72  7% Median Placebo 239.73 239.63  5%

Tesamorelin significantly reduced VAT area relative to placebo, with noeffect on SAT area (FIG. 2). Tesamorelin modestly increased lean bodymass by DXA, with no significant effect on total body fat mass (FIG. 2).Body mass index and waist circumference did not change.

Nutrition and Physical Activity

As assessed by Modifiable Activity Questionnaire, there were nosignificant changes in daily caloric and macronutrient intake byfour-day food record, self-reported alcoholic drinks per week, and hoursof activity per week between baseline and 12-months.

Sensitivity Analyses

Sensitivity analyses for the primary endpoint were performed usingmultiple imputation for missing data. This data confirmed the primaryresults, with estimated effect size of −3.8% (95% coverage −5.4, −2.2)reduction in HFF.

Adverse Effects

Adverse events by study group are shown in Table 4. A limited number ofserious adverse events (SAEs) were seen, which did not differ bytreatment arm. None were judged as related to study drug. In the placebogroup, two individuals had SAEs and were hospitalized: one following asuicide attempt, and one following a hematoma post liver biopsy. In thetesamorelin group, four individuals had SAEs and were hospitalized: onewith a history of stroke with transient hemiplegia for which a cause wasnot found, one due to suicidal ideation, one for urosepsis, and one forpneumonia and separately for influenza.

One individual in the placebo group and four in the tesamorelin grouphad events that met a priori protocol criteria for investigatordiscontinuation (P=0.17, including two discontinuations forhyperglycemia (2-week and 6-month visits) in the tesamorelin group. Onehad known diabetes at baseline. Other reasons for discontinuation areoutlined in FIG. 1.

TABLE 4 Adverse Events P-value for Tesamorelin Placebo comparison* Anyadverse event 29 29 0.57 Serious adverse event 4 2 0.41 Event meetingcriteria for 4 1 0.17 discontinuation by investigator Hyperglycemia 1211 Arthralgia 3 3 Myalgia 2 0 Paresthesia 2 2 Injection site bruising 1111 Injection site erythema 3 0 Injection site stinging 4 1 Otherinjection site complaints 10 1 URI 5 5 Other infection 7 12 Other 25 24Numbers refer to numbers of patients with events. *P-value forcomparison of numbers of events by group by Pearson Chi-Square. Thestudy was not powered to detect differences in adverse events, andp-values are shown only for aggregate events.

Example 3: Gene Set Enrichment Analysis (GSEA) on Samples fromTesamorelin and Placebo Groups

Gene Set Enrichment Analysis (GSEA) (Subramanian, Tamayo, et al., 2005,PNAS 102, 15545-15550; Mootha, Lindgren, et al., 2003, Nat Genet 34,267-273) using Hallmark and curated gene sets were performed on RNAseqdata generated liver tissues from Tesamorelin and Placebo groups todetermine whether Tesamorelin administration is associated with theoverexpression and/or downregulation of specific gene sets or pathways.Paired liver biopsy specimens from the study (tesamorelin, n=19;placebo, n=24) were utilized after preparation with RNAlater™. Change ingene expression was successfully evaluated in 18 patients in thetesamorelin group and 21 in the placebo group. An automated variant ofthe Illumina TruSeq™ Stranded mRNA Sample Preparation Kit was used todeliver 50M reads aligned in pairs. Each run is a 101 bp paired-end withan eight-base index barcode read. Data was analyzed using the BroadPicard Pipeline, which includes de-multiplexing and data aggregation.Alignment is completed using the STAR alignment algorithm. TruSeq StrandSpecific Large Insert RNA Sequencing includes plating, poly-A selectionand strand specific cDNA synthesis, library preparation (450-550 bpinsert size), sequencing (101 bp paired reads), sample identification QCcheck (when Sample Qualification of a matching DNA sample is chosen).The product provides library construction using a strand specificIllumina TruSeq™ Protocol and sequence coverage to 50M Paired reads or50M Total reads. Standardized gene set enrichment methodology was usedto compare changes in specific gene sets over time in each treatmentgroup. Data were analyzed using heat maps, enrichment plots, and dotplots for individual genes. Leading edge genes were examined in relevantpathways and in relationship to phenotypic changes on liver biopsy,including fibrosis stage.

Results from these studies are summarized below:

Cell Metabolism

-   -   Oxidative phosphorylation upregulated by treatment with        tesamorelin;    -   Leading edge genes correspond to genes that encode primarily:        -   Components of electron transport chain;        -   Chaperone proteins needed to assemble electron transport            chain;    -   Enhanced mitochondrial activity may reduce reactive oxygen        species (ROS) levels, preventing inflammation and fibrosis in        the liver.

Inflammation

-   -   Tesamorelin leads to a robust downregulation of multiple        inflammatory pathways. (e.g., TNF-alpha, IL-6 and IL-2);    -   Inflammation is an important process in NAFLD progression (e.g.,        NASH);    -   Gene analysis appears to be more sensitive to detect changes in        inflammation that were not reflected overtly in histopathology        analyses;    -   Leading edge genes correspond to genes that encode primarily:        -   Components of innate and adaptive immune cells;        -   Cytokines.

Tissue Repair

-   -   Tesamorelin led to a downregulation of genes involved in tissue        repair;    -   Fibrosis is a manifestation of a chronic/dysregulated wound        healing response. Notably, among tesamorelin-treated        participants, it was found that downregulation of gene pathways        involved in tissue repair corresponded to a reduction in genes        associated with fibrosis stage;    -   Apoptosis genes downregulated in tesamorelin-treated        participants;    -   UV response genes downregulated in tesamorelin-treated        participants;    -   Epithelial to mesenchymal transition (EMT) genes downregulated        in tesamorelin-treated participants;    -   Transforming growth factor-beta (TGF-β), a key mediator of        fibrosis, EMT, and tumorigenesis, was downregulated in        tesamorelin-treated participants.

Cell Turnover

-   -   Tesamorelin led to a downregulation of genes involved in cell        turnover;    -   Earliest event in NAFLD, even prior to steatosis, has been shown        to be liver proliferation; has been posited as an important step        in liver damage;    -   Repetitive cycles of apoptosis and proliferation of hepatocytes        increase the risks of aberrant repair in some individuals,        culminating in tumor initiation.

Cancer Prognosis Gene Sets

-   -   Tesamorelin led to upregulation of HCC good prognosis and        downregulation of HCC poor prognosis gene sets; downregulation        of Yap Taz pathway gene set, involved in fibrosis and        oncogenesis;    -   Consistent with its downregulation of inflammation, fibrogenic,        and cell turnover pathways, which together may culminate to        reduce malignancy risk    -   Tesamorelin appears to decrease hepatic malignancy potential,        even while agonizing the GH pathway and increasing systemic        IGF-1 pathways

Relationship of Specific Pathways to Changes in Fibrosis Genes (withinTesamorelin Group)

-   -   The gene set involved with oxidative phosphorylation negatively        related to change in fibrosis genes;    -   The gene sets involved in inflammation, tissue repair, cell        turner positively related to change in fibrosis genes.

Although the present disclosure has been described hereinabove by way ofspecific embodiments thereof, it can be modified, without departing fromthe spirit and nature of the subject disclosure as defined in theappended claims. In the claims, the word “comprising” is used as anopen-ended term, substantially equivalent to the phrase “including, butnot limited to”. The singular forms “a”, “an” and “the” includecorresponding plural references unless the context clearly dictatesotherwise.

What is claimed is:
 1. A method for (i) preventing or delaying the onsetof cirrhosis or (ii) treating cirrhosis, in a subject suffering fromnonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH),comprising administering an effective amount oftrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or a pharmaceutically acceptable saltthereof to said subject.
 2. The method of claim 1, wherein said subjecthas a hepatic fat fraction (HFF) of at least about 10%.
 3. The method ofclaim 1, wherein said subject has a hepatic fat fraction (HFF) of atleast about 15%.
 4. The method of claim 1, wherein said subject has ahepatic fat fraction (HFF) of at least about 20%.
 5. The method of claim1, wherein said subject has serum ALT levels of at least about 30 U/L.6. The method of claim 1, wherein said subject has serum ALT levels ofat least about 35 U/L.
 7. The method of claim 1, wherein said subjecthas a NAFLD Activity Score (NAS) of at least 1 as measured by the NASClinical Research Network (NAS CRN) scoring system.
 8. The method ofclaim 1, wherein said subject has a NAFLD Activity Score (NAS) of atleast 2 as measured by the NAS Clinical Research Network (NAS CRN)scoring system.
 9. The method of claim 1, wherein said subject has aNAFLD Activity Score (NAS) of at least 3 as measured by the NAS ClinicalResearch Network (NAS CRN) scoring system.
 10. The method of claim 1,wherein said subject has a NAFLD Activity Score (NAS) of at least 4 asmeasured by the NAS Clinical Research Network (NAS CRN) scoring system.11. The method of claim 1, wherein said subject has a reduction ofhepatic fat fraction (HFF) by at least 4% (absolute reduction) followingthe treatment.
 12. The method of claim 1, wherein said subject has liverfibrosis.
 13. The method of claim 12, wherein said subject has afibrosis score of at least 1C prior to the treatment.
 14. The method ofclaim 12, wherein said subject has a fibrosis score of at least 2 priorto the treatment.
 15. The method of claim 1, wherein said subjectsuffers from human immunodeficiency virus (HIV) infection.
 16. Themethod of claim 1, wherein said subject has a body mass index (BMI) ofat least about
 25. 17. The method of claim 1, wherein saidpharmaceutically acceptable salt of trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ isan acetate salt.
 18. The method of claim 1, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered by subcutaneous injection.
 19. The method ofclaim 1, wherein said trans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ orpharmaceutically acceptable salt thereof is present in a pharmaceuticalcomposition comprising at least one pharmaceutically acceptableexcipient.
 20. The method of claim 19, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in said pharmaceutical composition at a dose of about1 mg/ml to about 10 mg/ml.
 21. The method of claim 19, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in said pharmaceutical composition at a dose of about1 mg/ml to about 8 mg/ml.
 22. The method of claim 19, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is present in said pharmaceutical composition at a dose of about4 mg/ml to about 8 mg/ml.
 23. The method of claim 1, wherein saidtrans-3-hexenoyl-GHRH₍₁₋₄₄₎-NH₂ or pharmaceutically acceptable saltthereof is administered at a daily dose of about 1 mg to about 2 mg.